1 1 Symposium Chemosensory Receptors Satellite DEVELOPMENT ...

77 Poster Peripheral Olfaction and Peripheral TasteEXPRESSION OF GENES INVOLVED IN SYNTHESIS ANDSECRETION OF BIOGENIC AMINE NEUROTRANSMITTERSIN MOUSE TASTE BUDSDvoriantchikov G. 1 , Chaudhari N. 1 1 Physiology & Biophysics,University of Miami, Miami, FLCells within mammalian taste buds are known to express a number ofneurotransmitters. These may play roles in communication betweencells of a taste bud and/or for signaling to afferent nerve fibers. One ofthe biogenic amines, serotonin, is found in a subset of taste cells inmany species, and is released following taste stimulation (Huang et al.,2005). We explored the expression of genes involved in the synthesis ofbiogenic amine neurotransmitters in mouse taste buds. Aromatic L-amino acid decarboxylase (AADC) is common to the pathways forserotonin, dopamine, norepinephrine and epinephrine. We find thisenzyme is expressed at high concentration in taste buds, and is lackingfrom surrounding non-taste epithelium. Further, mRNA for tryptophanhydroxylase isoforms (Tph1 and 2) and the serotonin transporter all keyfor serotonin synthesis or packaging are also detected, albeit at lowerlevels. Enzymes involves in catecholamine synthesis, packaging orrelease, including Tyrosine Hydroxylase (TH), Vesicular MonoamineTransporter (VMaT2), Dopamine transporter, and α-sinuclein also areexpressed. Single-cell RT-PCR and immunocytochemical analysessuggest that some of these enzymes are found in a common subset ofcells in mouse taste buds. To date, we have been unable to detect eitherdopamine β-hydroxylase (DBH) or phenylethanola-mine-Nmethyltransferase(PNMT), enzymes necessary for the synthesis ofnorepinephrine and epinephrine. (Supported by DC006308 to NC).79 Poster Peripheral Olfaction and Peripheral TasteECTO-ATPASE IN TASTE BUDS OF FISHESKirino M. 1 , Kiyohara S. 1 , Hansen A. 2 , Finger T.E. 2 1 Chemistry andBioScience, Fac. Science, Kagoshima Univ., Kagoshima, Kagoshima,Japan; 2 Cell and Developmental Biology, University of ColoradoHealth Sciences Center, Aurora, COATP is claimed to be a crucial neurotransmitter in mammalian tastebuds (Finger et al., Science 2005) and taste buds in mammals displayrobust ectoATPase activity, suggesting that this enzyme plays a role ininactivation of the neurotransmitter. We utilized histochemicaltechniques to assess whether ectoATPase activity is present in tastebuds of fishes. Taste bud-bearing tissue was obtained from 3 species offish: 2 catfish: Plotosus lineatus (barbels), Ictalurus punctatus (barbels),and goldfish, Carassius auratus (lips and palatal organ). Tissue wasfixed 1 hr in mixed aldehydes in Tris-maleate buffer, then washed andcryoprotected overnight. Free-floating sections were then placed inreaction solution (2 mM Pb(NO 3 ) 2 containing inhibitors of non-specificphosphatases (levamisole, ouabain, α,β-methylene ADP) along witheither ATP or ADP as substrates. Following a reaction period of 30min., the tissue was washed and exposed to 1% Ammonium sulfide for1 min. Reaction product outlined elongate cells of taste buds in alltissues treated with ATP but not ADP. This high specificity for ATP issimilar to the ectoATPase in rodent taste buds. Reaction product alsowas evident in the nerve bundles coursing through the submucosa andreaching the base of the taste bud. This neural ATPase, however, waspresent in tissue reacted with either ADP and ATP indicating thepresence of a different ATPase than is present in taste buds. Furtherstudies will determine the cell type or types that exhibit this ectoATPasereactivity. Supported by NIH Grant R01 DC007495 (T.E.F.)80 Poster Peripheral Olfaction and Peripheral Taste78 Poster Peripheral Olfaction and Peripheral TasteCELL-TO-CELL COMMUNICATION IN TASTE BUDS: THEROLE OF ATP AND 5HTHuang Y. 1 , Maruyama Y. 1 , Pereira E. 1 , Roper S.D. 1 1 Physiology &Biophysics, Miller School of Medicine, University of Miami, Miami, FLWe previously have shown that sweet, sour, and bitter stimuli, andK + -induced depolarization all evoke 5HT release from mouse vallatetaste buds in vitro (Huang et al 2005, J Neurosci 25:843). Furthermore,applying ATP also stimulates 5HT release. Procedures that interferewith ATP stimulation, including applying suramin (a purinoceptorantagonist) or apyrase (an ATPase), block taste-evoked 5HT release.These data suggest that ATP acts as an intermediary in the release of5HT from taste buds. Here we present direct evidence for ATP releasefrom taste cells. Using CHO cells expressing highly sensitive P 2Xreceptors as biosensors, we are able to detect ATP release from isolatedtaste buds. ATP release was evoked by saccharin (2 mM), SC45647(100 µM), cycloheximide (10 µM), or denatonium (1 mM). We nextisolated single vallate taste cells, loaded them with Fura-2 for Ca 2+imaging, and identified 2 separate cell populations—cells stimulated bytastants (taste receptor cells) and cells stimulated by depolarization(synaptic cells; DeFazio et al, AChemS 2005). Isolated taste receptorcells released ATP and isolated synaptic cells released 5HT in biosensorstudies. There was zero overlap in these two populations. Our dataemphasize the existence of two functional classes of taste cells (cf.DeFazio et al, ibid., and Kim et al, this meeting) and indicate thatmouse vallate taste receptor cells communicate with nearbyserotonergic synaptic taste cells via ATP. Specifically, ATP secretedfrom receptor cells triggers 5HT release. (Supported by DC007630[SDR])NUMERICAL DENSITY OF TASTE CELLS IN RAT ANDMOUSE CIRCUMVALLATE TASTE BUDSMa H. 1 , Yang R. 1 , Kinnamon J.C. 1 1 Biological Sciences, University ofDenver, Denver, COWe have previously shown that significant differences exist betweenmouse and rat taste buds with regard to the expression of markers fortaste signaling and synaptic proteins (Ma et al., AChemS 2005). In thepresent study we wished to determine if there are differences in thenumerical density of taste cells and taste bud volume between rat andmouse taste buds. For this study we sliced serial transverse sections (1µm thickness) from mouse and rat circumvallate papillae to count thenumbers of taste cells in the taste buds and to calculate taste budvolume. Forty-one taste buds from 3 mice and 42 taste buds from 3 ratswere analyzed. Our results indicate that mouse taste buds contain anaverage of 85.8 taste cells vs 68.4 taste cells in rat taste buds. Thenumbers of taste cells/bud ranged from 32-152 in the mouse and 34-126in the rat. Although the average mouse taste bud contains more tastecells than a rat taste bud, the average volume of a mouse taste bud(42,000 µm 3 ) is smaller than a rat taste bud (64,200 µm 3 ). Thenumerical density of taste cells in mouse circumvallate taste buds (2.1cells/1000 µm 3 ) is significantly higher than that in the rat (1.2cells/1000 µm 3 ). The results of the present study, taken together withour previous work, demonstrate that mouse and rat taste buds are bothquantitatively and qualitatively different. This work is funded by NIHgrant DC00285.20